Silenced chromatin is permissive to activator binding and PIC recruitment.

Chromatin is thought to repress transcription by limiting access of the DNA to transcription factors. Using a yeast heat shock gene flanked by mating-type silencers as a model system, we find that repressive, SIR-generated heterochromatin is permissive to the constitutive binding of an activator, HSF, and two components of the preinitiation complex (PIC), TBP and Pol II. These factors cohabitate the promoter with Sir silencing proteins and deacetylated nucleosomal histones. The heterochromatic HMRa1 promoter is also occupied by TBP and Pol II, suggesting that SIR regulates gene expression not by restricting factor access to DNA but rather by blocking a step downstream of PIC recruitment. Interestingly, activation of silent promoter chromatin occurs in the absence of histone displacement and without change in histone acetylation state.

Chromatin and cancer: causes and consequences.

In this review, we discuss recent evidence implicating chromatin structure in the etiology of cancer. In particular, we present evidence indicating that inappropriate regulation of chromatin structure inhibits normal cell differentiation pathways and stimulates uncontrolled cell proliferation, with the outcome being oncogenesis. Such inappropriate chromatin structures arise as a consequence of (i) chromosomal rearrangements that fuse gene-specific activators with global co-regulators, drastically altering activator function; (ii) hypermethylation of tumor suppressor gene promoters, resulting in their inactivation; or (iii) mistargeted nuclear compartmentalization of growth-control genes and their regulators, resulting in the up- or down-regulation of such genes. How does chromatin silence genes? Recent results from model in vivo systems argues that chromatin can repress transcription at two levels: (i) by sterically interfering with the binding of transcription factors to the promoter, thereby blocking initiation; and (ii) at a step subsequent to the binding of activators and recruitment of the preinitiation complex. J. Cell. Biochem. Suppl. 35:61-68, 2000.

SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin.

Previous work has suggested that products of the Saccharomyces cerevisiae Silent Information Regulator (SIR) genes form a complex with histones, nucleated by cis-acting silencers or telomeres, which represses transcription in a position-dependent but sequence-independent fashion. While it is generally thought that this Sir complex works through the establishment of heterochromatin, it is unclear how this structure blocks transcription while remaining fully permissive to other genetic processes such as recombination or integration. Here we examine the molecular determinants underlying the silencing of HSP82, a transcriptionally potent, stress-inducible gene. We find that HSP82 is efficiently silenced in a SIR-dependent fashion, but only when HMRE mating-type silencers are configured both 5' and 3' of the gene. Accompanying dominant repression are novel wrapped chromatin structures within both core and upstream promoter regions. Strikingly, DNase I footprints mapping to the binding sites for heat shock factor (HSF) and TATA-binding protein (TBP) are strengthened and broadened, while groove-specific interactions, as detected by dimethyl sulfate, are diminished. Our data are consistent with a model for SIR repression whereby transcriptional activators gain access to their cognate sites but are rendered unproductive by a co-existing heterochromatic complex.

Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro.

Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeast HSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced approximately 100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant HSF for the full-length HSP82 promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels. We propose that the remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites.